Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965–2020

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965–2020. The PenNS proportion increased from 0.3% in 1965–1985 to 7.0% in 2005–2014 and to 33.3% in 2015–2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

N. meningitidis can be divided into 12 serogroups, and evolutionary relationships among isolates from within and without serogroup can be described by clonal complex (CC), defined by multilocus sequence typing (MLST) (2). The distribution of serogroups and CCs varies by time and geographic location.
In the past 20 years in China, N. meningitidis serogroup C (NmC) CC4821 has replaced N. meningitidis serogroup A (NmA) CC5 as being predominant nationwide (3)(4)(5)(6). This replacement was driven by national dissemination of a hyperinvasive and quinolone-resistant clone within CC4821, China CC4821-R1-C/B , and led to the high frequency of resistance (≈70%) of meningococci in China against fluoroquinolones, which had been used as antimicrobial prophylaxis for close contacts of IMD patients since 2005 (5).
CC4821 is expanding worldwide and has been found in 19 countries outside of China (7); infections include urogenital and anorectal infections among men who have sex with men in Europe (8). Global CC4821 diverges into 4 sublineages, of which a high proportion (79.3%) of CC4821 isolates in Europe and in North and South America possess molecular markers of nonsusceptibility to penicillin (Pen NS ). In contrast, the proportion was much lower in China (10.5%) (7).
In several countries, the first-line therapeutic antimicrobial therapies for IMD have been penicillin and third-generation cephalosporins (3GCs), such as cefotaxime and ceftriaxone (9); long-term meningococcal chemoprophylaxis for patients using complement inhibitors includes penicillin (10). Because IMD can cause death within hours (11), the frequency of Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (Pen NS ) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The Pen NS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 Pen NS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three Pen NS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the Pen NS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.
infections with N. meningitidis resistant to penicillin and 3GCs is an issue of great concern worldwide. N. meningitidis resistance to 3GCs is rare, and only 1 cefotaxime-resistant isolate has been reported in the United Kingdom (12). In recent years, Pen NS meningococci have become more frequent worldwide (13,14), but data for meningococci from China with Pen NS and 3GCs resistance remain poorly described. Two (17). However, information regarding the resistance mechanism and the genetic origin is unavailable. On the basis of N. meningitidis and commensal Neisseria isolates in Shanghai, China, since 1965, our aim with this study was to report the proportion and clonal relationship of Pen NS isolates, demonstrate the origin and evolutionary changes of their penA genes, and evaluate the role of CC4821 in disseminating penicillin and 3GC resistance.

Isolate Collection
During 1965-2020, a total of 491 meningococcal and 724 commensal Neisseria isolates were collected in Shanghai. The meningococci were isolated from 171 IMD patients and 320 asymptomatic carriers during 1965-1985 and 2005-2020 (5), and the commensal Neisseria isolates were isolated from healthy persons during 2013 and 2019 (18).

Antimicrobial Susceptibility Testing
Using the agar-dilution method, we determined MICs of penicillin, azithromycin, cefotaxime, ceftriaxone, meropenem, chloramphenicol, ciprofloxacin, minocycline, rifampin, and trimethoprim/sulfamethoxazole. Using antibiotic gradient strip diffusion methods (Etest; bioMérieux, https://www.biomerieux.com), we determined the MICs for the Pen NS isolates and cefotaxime-resistant isolates. We interpreted breakpoints according to the 2022 guidelines of the Clinical and Laboratory Standards Institute (19).

N. meningitidis Isolate Typing
We determined the serogroup of N. meningitidis isolates by using slide agglutination with monoclonal antiserum (Remel Europe Ltd., www.remel.com). All isolates were analyzed by MLST and typing for PorA and FetA according to previously described protocols (3). We analyzed whole-genome sequences of the Pen NS meningococci by using the meningococcal coregenome MLST (cgMLST) schemes of N. meningitidis cgMLST version 1.0 for Pen NS meningococci and the L44 cgMLST schemes for CC4821 isolates (7,20).

Analysis of Penicillin and 3GC Resistance-Associated Genes
Low-level penicillin resistance and 3GC resistance of N. meningitidis are mainly associated with mutations in the penicillin-binding protein (PBP) 2, which can be determined by sequencing its coding gene, penA, using the primers recommended by Taha et al. (14). On the basis of a 402-bp fragment (nucleotides 1321-1722) encoding transpeptidase domain (14), we determined the penA alleles according to the nomenclature in the Neisseria PubMLST database (21). We submitted novel penA alleles discovered in this study, and they were assigned new allele numbers in the database. The ponA gene encoding PBP1, in which the mutation L421P was reportedly associated with penicillin resistance in N. gonorrhoeae, was analyzed as previously described (22). We performed phylogenetic analyses via maximum-likelihood analysis with IQ-TREE version 2.2.0 (23), using the 402-bp penA sequences collected in this study and those in the Neisseria PubMLST database from different Neisseria species and different countries, deposited before December 25, 2021 (21).

Determination of Potential Donors and Recombination Crossover Points of Meningococcal penA Alleles
On the basis of previously described criteria, we considered a commensal Neisseria strain to be a potential donor for a recombinant penA allele in N. meningitidis (18). To identify the donors and the crossover points, we performed Illumina sequencing (https://www. illumina.com) on representative N. meningitidis and commensal Neisseria isolates that shared a candidate recombinant penA allele. We checked combination crossover points identified by visual inspection by using RDP software (Recombination Detection Program), version 4.97 (24).

Genetic Transformation
We performed the transformation of chromosomal DNA (500 ng) and penA fragment (100 ng) from Neisseria donor isolates into N. meningitidis as previously described (18). We selected 3 transformants of each pair of donor and recipient isolates for further study. We determined the penicillin MICs by using Etest and the genomes of the transformants by using Illumina sequencing. We submitted the genomes of N. meningitidis and commensal Neisseria that were sequenced in this study to PubMLST Neisseria Database with PubMLST identification numbers (Appendix 1 Table 1, https:// wwwnc.cdc.gov/EID/article/29/2/22-1066-App1.xlsx).

Phylogenetic Analysis of penA Alleles of Pen NS Meningococci
To track the genetic origin of penA alleles of the Pen NS isolates, we also analyzed the penA nucleotides of 724 commensal Neisseria isolates collected during 2013-2019. We found 288 penA alleles, all with the 5 common amino acid mutations in PBP2. The allele penA795 was the most frequent (8.6%, 62/724), followed by penA964 (4.7%, 34/724) and penA808 (4.4%, 32/724).
Among the 20 penA alleles from the Shanghai Pen NS meningococci, only penA184 (A549T) was within the N. meningitidis cluster; the other 19 alleles (with >2 of the 5 mutations) scattered into the N. lactamica cluster (n = 7), the N. subflava cluster (n = 3), the N. gonorrhoeae cluster (n = 1), or outside the 5 clusters (n = 8). Those findings suggest that the PBP2 for the mutations was acquired by horizontal gene transfer ( Figure 3).

Crossover Point of Recombination Events in penA
Among the 19 Pen NS meningococcal penA alleles acquired by horizontal gene transfer, we found that 6 penA alleles (penA110, penA405, penA552, penA795, penA832, and penA843) were shared by N. meningitidis and commensal Neisseria isolates (Appendix 2 Table  2). We analyzed 47 Neisseria genomes harboring these 6 alleles and found all potential donors of the 6 penA alleles; the sizes of the recombination fragments were 805-2,491 bp (Appendix 2 Table 2).
We discovered that the penA795 allele, an allele associated with dual resistance to penicillin and 3GCs, was also harbored in the internationally disseminated ceftriaxone-resistant N. gonorrhoeae FC428 clone (Appendix 2 Table 2) (25). It was difficult to judge the origin donor of penA795 because it was outside all the phylogenetic clusters and shared by 6 species of Neisseria (Appendix 2 Table 2).
Ceftriaxone is structurally similar to cefotaxime, sharing an exact R1 side chain and similar molecular mechanisms of action. We identified several mutations in the C-terminal or transpeptidase domain of the mosaic PBP2 that were associated with reduced cephalosporin susceptibility (e.g., I312M, V316T, F504L, N512Y, and G545S) (31); mutations A311V and T483S were associated with conferring ceftriaxoneresistance to N. gonorrhoeae. The cefotaxime-resistant meningococci from China (that contained mutations A311V and T483S in PBP2) differed from the cefotaxime-resistant isolate from the United Kingdom (with PBP2 mutations A501T and D511V) (12) but were consistent with the internationally disseminated ceftriaxone-resistant N. gonorrhoeae FC428 clone (25). , and other commensal Neisseria (n = 73) isolates and genomes collected in this study and from the Neisseria PubMLST database was constructed by using IQ-TREE version 2.2.0 (23), with both SH-aLRT test and UFboot set as 1,000. The values of SH-aLRT and ultrafast bootstrap (Ufboot) are shown on the node of each clade as SH-aLRT/Ufboot. Clusters were determined by using SH-aLRT values of 80% from the SH-aLRT tests with 1,000 replicates and ultrafast bootstrap (UFBoot) values of 85% from bootstrap tests with 1,000 replicates (IQ-TREE). Alleles penA378, penA405, penA552, penA553, penA843, penA868, and penA917 were within in the N. lactamica cluster; penA662, penA777, and penA865 were within the N. subflava cluster; penA379 was within the N. gonorrhoeae cluster; and the other 8 penA alleles were located outside the 5 clusters. Scale bar indicates substitutions per site. Pen NS , penicillin-nonsusceptible meningococci.
Both penA795-bearing isolates Nm507 and FC428 had identical transpeptidase domains of PBP2 (Appendix 2 Figure 3), whereas FC428 displayed a 4-fold higher ceftriaxone MIC than Nm507, possibly resulting from the alterations in penB and the promoter region of mtrR in FC428 (32). We discovered that penA795 was predominant in commensal Neisseria isolates in Shanghai, disseminated among N. lactamica, N. cinerea, N. polysaccherae, N. subflava, and N. gonorrhoeae. Phylogenetic analysis and genetic transformation suggest that the meningococcal cefotaxime resistance probably originates from N. subflava (penA777 and penA865) and another unknown Neisseria (penA795).
As a hyperinvasive lineage first identified in the NmC meningococcal outbreaks in Anhui, China, dur-  (3,33). In 2015, a total of 79% of CC4821 isolates were reported to possess quinolone resistance, and it was recommended that ciprofloxacin not be used as chemoprophylaxis for IMD in 2021 (5,16). In our study, we observed an increasing trend for acquisition of penicillin nonsusceptibility and cefotaxime resistance in quinolone-resistant CC4821 isolates, which further narrowed the choices for antimicrobial treatment and prophylaxis; safe and effective alternatives such as ceftriaxone, rifampin, and azithromycin could be considered to deal with this hyperinvasive lineage. Another concern is that NmB has become dominant in penicillin-and quinolone-resistant strains, accompanied by increasing nongroupable or rare serogroups (such as NmY and NmW), which could not be protected at present by vaccines in the Expanded Programme for Immunization in China (33).
Widespread resistance to either penicillin or ciprofloxacin, which is often associated with emergence of new resistant clones, has challenged the local strategies for treating and preventing IMD. After 2016, a new penicillin-resistant clade of W:P1.5,2:F1-1 (CC11) expanded from Australia to Europe and North America (13,34). In 2021, a bla ROB-1 -containing Y:P1.5-2,10-2:F4-1:ST-3587(CC23) clone, which showed dual resistance to penicillin and ciprofloxacin, was identified in the United States and El Salvador (35,36). Among global CC4821 isolates, 2 antimicrobial-resistant clones were discovered: one is China CC4821-R1-C/B (quinolone-resistant, gyrA71, NmC and NmB), expanding from China to other countries, and the other is the Europe-USA CC4821 cluster (Pen NS , penA9, NmB), which was restricted to countries outside of China (7). In our study, we observed rapid increases of the Pen NS meningococcal strains with diversified Pen NS alleles, which should be attributable to the selective pressure of penicillin after the increased consumption of broad-spectrum penicillin as indicated by the genetic diversity of these strains. Of note, 42.3% of the Pen NS isolates and 2/3 of the cefotaximeresistant isolates were assigned to the CC4821 cluster ( Figure 2). Among the 11 Pen NS isolates in the CC4821 cluster, 7 isolates were assigned to the same sublineage, L44.2. Among the L44.1 sublineage (identical to the hyperinvasive epidemic clone, China CC4821-R1-C/B ), penicillin-, cefotaxime-, and quinolone-resistant ST-4821 strains have caused IMD, which should raise more concerns for public health.
Most (25/26) of the Pen NS isolates in our study (Appendix 1 Table 2) are not covered by the scheduled meningococcal vaccines (MPV-A and MPV-AC) in China (33). All 3 cefotaxime-resistant isolates were from toddlers, who were unable to obtain protection from the corresponding vaccines (NmC or NmB) according to scheduled vaccination in China (33). MPV-AC is used only for children >3 years of age, and no NmB vaccines are available nationwide. To protect young children from cefotaxime-resistant isolates, on one hand, serogroup A and C meningococcal polysaccharide conjugate vaccine could be a good choice because it can cover populations >3 months of age (37); on the other hand, it is necessary to introduce or develop NmB vaccines for CC4821 strains from China.
In our study, 18/20 penA alleles identified in the Pen NS isolates harbored the 5 penicillin-resistanceassociated mutations in PBP2, and no prevalent alleles were found. In Europe and the United States, penA12 (8%), penA14 (6%), and penA9 (5%) were the most prevalent alleles in Pen NS isolates (14), but none of them were observed in isolates from China.
Phylogenetic analysis showed that most of the altered penA fragments of the Pen NS isolates from China were acquired by horizontal gene transfer and most likely from N. lactamica, N. subflava, and N. gonorrhoeae. Analysis of >700 local commensal Neisseria isolates showed that their PBP2 all harbored the 5 common mutations, which could provide N. meningitidis isolates with various mutation-harboring penA alleles. On the basis of 6 penA alleles shared by N. meningitidis and commensal Neisseria isolates, those potential horizontal gene transfer events were validated by sequence analysis and genetic transformation (Appendix 2 Tables 2, 3).
One limitation of this study is the limited number of IMD isolates, which is mainly attributable to the recent low and decreasing incidence of IMD in China, from 0.18 cases (2005) to 0.0078 cases (2015-2019) per 100,000 population (33,38). Nevertheless, the isolates were phylogenetically related to the invasive meningococci in China, possessing representative features as demonstrated previously (5,7,39,40). The trend of increasing Pen NS meningococci in China provided additional evidence for this study (15)(16)(17). Another limitation is that the penA184 and penA866 alleles were represented by only 1 penicillin-intermediate isolate each, which did not meet the requirements for the definition of Pen NS penA alleles (14), although genetic transformations supported the contributions to the phenotype.
In summary, our study detected an ongoing shift in the penicillin susceptibility of meningococcal isolates in Shanghai. Pen NS meningococcal isolates have increased in recent years, and Pen NS CC4821 isolates have become predominant. Resistant penA alleles have been captured by quinolone-resistant CC4821 hyperinvasive epidemic clone with serogroup B or C. Because we do not yet have NmB vaccines with high coverage for NmB isolates of China CC4821-R1-C/B , the concern is that the triple-resistant CC4821 clone has the potential to cause an epidemic. The altered penA of Pen NS isolates originated mainly from commensal Neisseria isolates, including N. lactamica and N. subflava. As part of the increasing trend of penicillin nonsusceptibility among N. meningitidis isolates in China during 1965-2020, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone.